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1 State Key Laboratory of Microbial Resources, Institute of Microbiology, Chinese Academy of Sciences, No. 13, North 1st Ave, Zhong Guan Cun, Beijing 100080, PR China
2 Graduate School, Chinese Academy of Sciences, Beijing 100080, PR China
Correspondence
Xiuzhu Dong
dongxz{at}sun.im.ac.cn
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5c (16·63 %) and isoC17 : 1 I (15·34 %). LL-Diaminopimelic acid existed in the cellular peptidoglycan. The genomic DNA G+C content of strain GB4-38T was 43·2 mol%. Phylogenetic analysis based on 16S rRNA gene sequences supported clustering of the two strains with syntrophic bacterial species of the genus Syntrophomonas (89·6–92·4 % sequence similarity), but phenotypic, chemotaxonomic and genetic characters differentiated the two strains from members of this genus. Therefore, it is proposed that the two strains are representatives of a novel species, Syntrophomonas erecta sp. nov. The type strain is GB4-38T (=CGMCC 1.5013T=DSM 16215T).
Published online ahead of print on 29 October 2004 as DOI 10.1099/ijs.0.63372-0.
The GenBank/EMBL/DDBJ accession numbers for the 16S rRNA gene sequences of Syntrophomonas erecta strains GB4-38T and SB9-1 are AY536889 and AY643536, respectively.
Electron micrographs of GB4-38T cells are available as supplementary material in IJSEM Online.
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). So far, eight species of syntrophic anaerobes have been described that oxidize SCFAs with more than 4 carbon atoms and long-chain fatty acids of up to 18 carbon atoms via synergetic metabolism with hydrogen-consuming partners (McInerney et al., 1979, 1981; Stieb & Schink, 1985; Roy et al., 1986; Lorowitz et al., 1989; Zhao et al., 1990; Svetlitshnyi et al., 1996; Jackson et al., 1999; Sekiguchi et al., 2000; Zhang et al., 2004). Physiological characterization and phylogenetic analysis clustered these syntrophic bacteria into two lineages: the family Syntrophomonadaceae (McInerney, 1992; Zhao et al., 1993), within the low-G+C-containing Gram-positive bacteria, and the family ‘Syntrophaceae’ in the subclass 
-Proteobacteria (Jackson et al., 1999). In the present study, two novel syntrophic bacterial strains, GB4-38T and SB9-1, are described. They degraded straight-chain fatty acids with 4–8 carbon atoms into propionate, acetate and methane in co-culture with a methanogen. 16S rRNA gene sequence analysis indicated their affiliation to the family Syntrophomonadaceae; however, morphological, chemotaxonomic and genetic characters distinguished them from known species. Therefore, a novel species of Syntrophomonas is proposed.
Methanospirillum hungatei DSM 864T was kindly provided by A. Stams (Department of Microbiology, Wageningen University, The Netherlands). Syntrophomonas wolfei subsp. wolfei DSM 2245BT and Syntrophomonas sapovorans DSM 3441T were purchased from the DSMZ (Braunschweig, Germany). Syntrophomonas curvata DSM 15682T was preserved in our laboratory. Strain GB4-38T was isolated from the granular sludge of an upflow anaerobic sludge blanket (UASB) reactor for treating wastewater from a bean-curd farm in Beijing, China, and strain SB9-1 was isolated from lotus-growing mud in a Beijing suburb.
A pre-reduced basal medium described by McInerney et al. (1979) was used for isolation and routine cultivation. The gas phase was normally N2/CO2 (80 : 20; 1·01x105 Pa), although H2/CO2 (80 : 20; 1·25x105 Pa) was used for cultivation of M. hungatei DSM 864T. Inoculation and transfer of the cultures were done with a syringe and needle. The purity of strain GB4-38T in co-culture and pure culture was examined periodically by monitoring cell morphology using normal bright field microscopy and colonies on solid media, as well as the absence of growth in a rich medium like peptone-yeast extract-glucose (PYG) liquid.
Substrate utilization and potential electron acceptors for butyrate degradation were examined according to Zhang et al. (2004). Cell growth was monitored by determining the OD600 of the culture. Acetate, propionate and methane production and substrate degradation were measured by GC (GC-14A/B; Shimadzu) and HPLC (series 1050; Hewlett Packard) as previously described (Touzel & Albagnac, 1983; Jackson et al., 1999; Zhang et al., 2004). The molar growth yield of strain GB4-38T in pure culture was determined in 50 ml basal medium with 20 mM crotonate; growth in basal medium without crotonate was used as a control. Cell mass was obtained by centrifuging 72 h cultures at 7000 g and resuspending the cell pellet in 10 mM monobasic potassium phosphate buffer (pH 7·0). The cell suspension was dried at 100 °C overnight and dry cell mass was determined according to Jackson et al. (1999).
Exponential-phase cells of strain GB4-38T were used for morphology examination with a TEM (H-600A; Hitachi). Before observation, negative staining with uranyl acetate was performed. Ultrathin sections were stained with uranyl acetate and lead citrate according to Reynolds (1963).
Cellular fatty acids of strain GB4-38T were analysed as fatty acid methyl esters with the MIDI microbial identification system. Diamino acids of the cell wall were assayed using TLC on a cellulose plate and the solvent system of Rhuland et al. (1955).
Genomic DNA of strain GB4-38T grown on crotonate was extracted as previously described (Marmur, 1961). The G+C content was determined using the thermal denaturation method (Marmur & Doty, 1962) with a DU 800 spectrophotometer (Beckman). DNA from Escherichia coli K-12 was used as a reference for the thermal melting profile (Tm). DNA relatedness was determined on the basis of DNA–DNA liquid reassociation rate (De Ley et al., 1970) at 65 °C using a DU 800 spectrophotometer (Beckman).
The 16S rRNA gene was amplified by PCR and then sequenced using the method described by Weisburg et al. (1991). Sequences of GB4-38T, SB9-1 and reference strains (obtained from GenBank) were aligned using the program DNAMAN (version 4.0; Lynnon Biosoft). A phylogenetic tree was constructed by the neighbour-joining method and the topology of the tree was evaluated by bootstrap analysis of 1000 datasets with DNAMAN.
Methanogenic sludge samples were inoculated into pre-reduced basal medium containing 20 mM butyrate as sole carbon source. After subculturing several times in this medium, 5 % (v/v) enrichment culture and 5 % (v/v) M. hungatei DSM 864T culture were inoculated into the same medium with 1·6 % (w/v) agar and the Hungate roll-tube technique (Hungate, 1969) was performed. After repeating this procedure five to seven times, strains GB4-38T and SB9-1 were obtained, respectively, in co-culture with M. hungatei DSM 864T from the roll-tubes. 2-Bromoethanesulfonic acid (20 mM) was added to the co-cultures to inhibit the methanogen and, after subculturing 18 times, monocultures were obtained in the basal medium containing either 20 mM crotonate (GB4-38T) or 20 mM butyrate plus 20 mM pentenoate (SB9-1). Homogeneous cell morphology was observed in the monoculture. Without a methanogen, neither of the two strains degraded butyrate alone. However, the strains were able to resume butyrate degradation in artificial co-cultures with M. hungatei DSM 864T (data not shown). Hence, the two strains could be confirmed as syntrophic bacteria. In this study, a monoculture of S. wolfei subsp. wolfei DSM 2245BT was also obtained in a dozen transfers on crotonate medium containing 2-bromoethanesulfonic acid, i.e. in the absence of M. hungatei DSM 864T.
Cells of GB4-38T and SB9-1 were Gram-negative straight rods with round ends. They were 0·6–0·8 µm in width, 2·0–8·0 µm in length and occurred singly, in pairs or in chains. Spores were never observed. Electron microscopy showed the presence of multiple flagella interspersed around the subpoles of GB4-38T cells (micrograph available as supplementary material in IJSEM Online). Ultrathin sections of GB4-38T revealed the atypical Gram-positive cell wall structure (micrograph available as supplementary material in IJSEM Online). The two strains formed small, round, yellow colonies on butyrate medium in co-culture with M. hungatei DSM 864T.
Both GB4-38T and SB9-1 could obtain energy from the oxidation of straight-chain fatty acids with 4–8 carbon atoms in co-culture with M. hungatei DSM 864T. Benzoate, fatty acids longer than C8 and branched-chain fatty acids such as isobutyrate and isovalerate could not be utilized by the two strains (Table 1). Among the utilized substrates, butyrate, caproate and caprylate were degraded into acetate and presumably H2 (converted into CH4 by the methanogen) within 2 weeks, whereas valerate and heptanoate were converted into propionate, acetate and H2, which implied 
-oxidation of fatty acids used by the strains. When 1 mol butyrate was degraded, about 2 mol acetate and 2 mol hydrogen (converted into CH4) were produced; the carbon and electron recoveries were 97·76±4·18 % and 99·00±4·21 %, respectively. None of the following tested substances (all at 20 mM) could be used by the two strains as a potential electron acceptor in butyrate oxidization: sodium sulfate, sodium thiosulfate, sulfur, sodium nitrate and sodium fumarate.
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). Strain SB9-1 did not grow on crotonate. Growth and acid formation were not observed for the two strains on the following substrates: yeast extract (0·5 %), tryptone (1 %), peptone (1 %), maltose (10 mM), glucose (20 mM), fructose (20 mM), ribose (20 mM), xylose (20 mM), pentenoate (20 mM), fumarate (20 mM) and pyruvate (20 mM).
Temperature ranges for growth of GB4-38T in both co-culture on butyrate and pure culture on crotonate were 25–47 °C, with optimal growth at 37–40 °C. The pH range for growth was 6·0–8·8, with optimal growth at pH 7·8. The NaCl concentration range tolerated was 0–500 mM (optimal growth occurred below 50 mM NaCl). The profiles of temperature, pH and NaCl concentration for SB9-1 both in co-culture on butyrate and in pure culture on butyrate plus pentenoate were 25–45 °C (optimum at 37–40 °C), pH 6·7–9·0 (optimum at pH 7·5–8·5) and 0–500 mM NaCl (optimum below 50 mM NaCl). For both strains, no growth was observed in either pure culture or co-culture using air as the gas phase. The growth characteristics of S. wolfei subsp. wolfei DSM 2245T were retested and are shown in Table 1
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The major cellular fatty acids of strain GB4-38T were C14 : 0 (29·74 %), C16 : 0 (17·00 %), C16 : 15c (16·63 %) and isoC17 : 1 I (15·34 %), whereas C16 : 17c (38 %), C16 : 0 (16 %), C16 : 19c (16 %) and C14 : 0 (12 %) were predominant in S. wolfei subsp. wolfei DSM 2245T (Henson et al., 1988; Lorowitz et al., 1989) and C14 : 0 (27·82 %), C15 : 0 (19·24 %) and C14 : 0 3-OH (10·90 %), as well as an unknown component with an equivalent chain-length of 14·503 (21·44 %), constituted the main fatty acids of S. curvata DSM 15682T (Zhang et al., 2004). The peptidoglycan of GB4-38T contained LL-diaminopimelic acid, whereas S. wolfei subsp. wolfei DSM 2245T contained meso-diaminopimelic acid (McInerney et al., 1981; Lorowitz et al., 1989).
The 16S rRNA genes of strains GB4-38T and SB9-1 shared 98·8 % sequence similarity and these two strains had similar phenotypic characteristics, except for the substrate that supported the pure culture, indicating that the two strains could belong to one species. A phylogenetic tree including the two strains and other members of the family Syntrophomonadaceae was constructed (Fig. 1) and showed that the two strains clustered loosely with members of the genus Syntrophomonas. The 16S rRNA gene sequence similarities between strain GB4-38T and its three nearest relatives, S. curvata DSM 15682T, S. sapovorans DSM 3441T and S. wolfei subsp. wolfei DSM 2245T, were 92·4, 92·3 and 89·6 %, respectively.
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GB4-38T and SB9-1 also differed from other members of the genus Syntrophomonas in their phenotypic features. They were different from S. curvata DSM 15682T and S. sapovorans DSM 3441T in cell morphology, substrate range (Table 1), growth temperature and pH range and cellular fatty acid composition. Although our two strains had the same substrate range as S. wolfei subsp. wolfei DSM 2245T in co-culture, they could be distinguished from the latter by the following: growth at higher temperature and higher pH with different cell morphology (Table 1), diaminopimelic acid type in the peptidoglycan, cellular fatty acid composition and low DNA–DNA hybridization values. Based on multiphasic taxonomic traits, a novel syntrophic species, Syntrophomonas erecta sp. nov., is proposed.
Description of Syntrophomonas erecta sp. nov.
Syntrophomonas erecta (e.rec'ta. L. fem. adj. erecta erect).
Cells are straight rods with Gram-negative cell wall, 0·6–0·8x2·0–8·0 µm, non-spore-forming and with 2–5 flagella, usually occurring in the subpole of cells. Straight-chain fatty acids with 4–8 carbon atoms can serve as substrates for the species in co-culture with M. hungatei DSM 864T. Even-numbered fatty acids are degraded into acetate and presumably H2, whereas odd-numbered ones are degraded into propionate, acetate and H2. Straight-chain fatty acids shorter than C4 (acetate and propionate), longer than C8 (caprate, laurate, myristate, palmitate, stearate, oleate, linoleate and arachidate), branched-chain fatty acids (isobutyrate and isovalerate) and benzoate do not support the co-culture. Fumarate, sulfate, thiosulfate, sulfur and nitrate cannot act as electron acceptors for butyrate oxidation. Crotonate is the only tested substrate that supports growth of the type strain in pure culture, whereas butyrate plus pentenoate supports growth of SB9-1. Yeast extract, tryptone, peptone, maltose, glucose, fructose, ribose, xylose, pentenoate, fumarate and pyruvate alone do not support growth. The type strain can grow at pH 6·0–8·8, 25–47 °C and in 0–500 mM NaCl. The cellular fatty acids of the type strain contain mainly C14 : 0 (29·74 %), C16 : 0 (17·00 %), C16 : 15c (16·63 %) and isoC17 : 1 I (15·34 %). LL-Diaminopimelic acid exists in the cellular peptidoglycan. The G+C content of the genomic DNA is 43·2–43·9 mol%.
The type strain is GB4-38T (=CGMCC 1.5013T=DSM 16215T), isolated from the granular sludge of a UASB reactor for treating bean-curd farm wastewater in Beijing, China.
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ACKNOWLEDGEMENTS |
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